Farhat N, Matouk C C, Mamarbachi A M, Marsden P A, Allen B G, Thorin E
Department of Biochemistry, University of Montreal, Montreal, Quebec, Canada.
Br J Pharmacol. 2008 Apr;153(7):1420-31. doi: 10.1038/bjp.2008.25. Epub 2008 Feb 18.
The factors that influence the cellular levels of endothelin-1 (ET-1) include transcription, mRNA localization, stability and translation, post-translational maturation of preproET-1 and degradation of ET-1. We investigated the regulation of ET-1 mRNA abundance by extracellular ET-1 in porcine aortic endothelial cells (PAECs).
Passsage one cultures of PAECs were incubated in starving medium in the presence or absence of ET-1 and antagonists or pharmacological inhibitors. PreproET-1 mRNA, endothelin-1 promoter activity, Erk and p38 MAPK activation were determined.
Exogenous ET-1 reduced cellular ET-1 mRNA content: a reduction of 10 000-fold was observed after 4 h. ET-1 simultaneously reduced the stability of ET-1 mRNA and increased the loading of RNA Polymerase II at the endothelin-1 promoter. In the absence of exogenous ET-1, the ETB-selective antagonist, BQ788, increased ET-1 mRNA. An ETA-selective antagonist had no effect. ET-1 mRNA returned to control levels within 24 h. Whereas activation of p38 MAPK induced by ET-1 peaked at 30 min and returned to control levels within 90 min, Erk1/2 remained active after 4 h of stimulation. Inhibition of p38 MAPK prevented the ET-1-induced decrease in ET-1 mRNA. In contrast, Erk1/2 inhibition increased ET-1 mRNA. Similarly, inhibition of receptor internalization increased ET-1 mRNA in the presence or absence of exogenous ET-1.
These results suggest that extracellular ET-1 regulates the abundance of ET-1 mRNA in PAECs, in an ETB receptor-dependent manner, by modulating both mRNA stability and transcription via mechanisms involving receptor endocytosis and both ERK and p38 MAPK pathways.
影响内皮素 -1（ET-1）细胞水平的因素包括转录、mRNA定位、稳定性和翻译、前内皮素原（preproET-1）的翻译后成熟以及ET-1的降解。我们研究了细胞外ET-1对猪主动脉内皮细胞（PAECs）中ET-1 mRNA丰度的调节作用。
将传代一代的PAECs培养物在饥饿培养基中培养，分别加入或不加入ET-1、拮抗剂或药理抑制剂。测定前内皮素原mRNA、内皮素 -1启动子活性、Erk和p38丝裂原活化蛋白激酶（MAPK）的激活情况。
外源性ET-1降低了细胞内ET-1 mRNA含量：4小时后观察到降低了10000倍。ET-1同时降低了ET-1 mRNA的稳定性，并增加了RNA聚合酶II在内皮素 -1启动子上的结合。在没有外源性ET-1的情况下，ETB选择性拮抗剂BQ788增加了ET-1 mRNA。ETA选择性拮抗剂则无作用。ET-1 mRNA在24小时内恢复到对照水平。ET-1诱导的p38 MAPK激活在30分钟时达到峰值，并在90分钟内恢复到对照水平，而Erk1/2在刺激4小时后仍保持活性。抑制p38 MAPK可防止ET-1诱导的ET-1 mRNA减少。相反，抑制Erk1/2则增加了ET-1 mRNA。同样，在有或没有外源性ET-1的情况下，抑制受体内化均增加了ET-1 mRNA。
这些结果表明，细胞外ET-1通过涉及受体内吞作用以及ERK和p38 MAPK途径的机制，以内皮素B（ETB）受体依赖性方式调节PAECs中ET-1 mRNA的丰度，同时调节mRNA稳定性和转录。
